Rotaviruses, a large and genetically diverse group of segmented double-stranded (ds)RNA viruses, are a significant cause of gastroenteritis in many mammalian and avian species. Among infants and young children, rotavirus infections can result in life-threatening dehydrating diarrheal disease. Like other RNA viruses with segmented genomes, rotaviruses have the potential to evolve rapidly through exchange of genome segments (reassortment) during co-infection. Although novel reassortant virus strains occasionally infect humans, most human disease is caused by rotaviruses possessing either of two preferred genome constellations. Why the two preferred constellations are maintained is not clear, but may stem from the long term co-evolution of human rotavirus genome segments giving rise to sets of viral RNAs and/or proteins that function optimally when maintained together. One of the single greatest mysteries that remain about rotaviruses, and indeed all members of the Reoviridae family, is how their RNAs are selectively recognized and packaged during the assembly of progeny particles, a process that must be highly specific to assure that newly made virions end up with a complete constellation of genome segments. With the recent development of a fully plasmid-based rotavirus reverse genetics (RG) system, it is now possible to characterize the rotavirus assortment process, including dissecting the structure and function of genetic determinants needed for segment-specific RNA recognition and investigating the role of viral and hosts proteins in promoting and chaperoning the process. It is the goal of this grant proposal to pursue such studies, which will not only reveal the molecular basis of rotavirus genome assortment, but also may reveal how assortment is connected to the maintenance of preferred genome constellations. Specifically, the Aims of this grant application are as follows: (i) Elucidate the location and properties of genetic determinants that are required for segment-specific recognition and packaging (assortment) of viral RNAs into progeny particles. (2) Delineate the importance of the viral and host RNA-binding proteins in promoting and chaperoning segment-specific recognition and packaging of viral RNAs. These aims will be addressed using the rotavirus RG system in combination with complementary methods developed in our laboratory that have allowed us to map cis-acting replication in rotavirus RNAs and analyze the specificity and targets of rotavirus RNA binding proteins. By accomplishing these Aims, it may be possible to manipulate packaging signals in rotavirus vaccine candidates in such a manner as to prevent their reassortment with naturally circulating rotavirus stains.